Engineering polypeptide folding through trans … polypeptide folding through trans double bonds: Transformation ... Symmetry transformations used to generate equivalent atoms

S1

Engineering polypeptide folding through trans double bonds: Transformation

of miniature ββββ-meanders to hybrid helices

Mothukuri Ganesh Kumar,a Sushil N. Benke,a K. Muruga Poopathi Raja*b and Hosahudya N. Gopi*a

a Department of Chemistry, Indain Institute of Science Education and Research, Dr. Homi Bhabha Road, Pune-411 008, India

b Department of Physical Chemistry, School of Chemistry, Madurai Kamaraj University, Madurai-625 021, India

*Corresponding authors: [email protected]; [email protected]

List of Contents

1. ORTEP diagrams of peptides P1, P3 and P4.…..………………...…………….….…....S2

2. Hydrogen bonding parameters for peptidesP1, P3 and P4…………..…………….…...S6

i) Intra-molecular hydrogen bonding parameters

ii) Intermolecular hydrogen bonding parameters

3. Torsion angle tables for peptides P1, P3 and P4…..……………………...……………S10

4. Crystallographic information of peptides P1,P3 and P4……...…………………….….S11

5. General experimental details…………………………………………………………...S13

6. 2D NMR data of peptides P1 and P2…….....………………………………………….S14

i) List of NOE’s of peptides

ii) ROESY and TOCSY spectra of peptides

iii) Schematic representation of observed NOEs

7. Synthesis and characterization of Boc-dγF-OEt,Boc-dγA-OEtand peptides ……........S22

8. CD spectra of peptides………………………………………………………………….S26

9. HPLC traces of peptides…………………………………………………………….…S29

10. Variable Temperature 1H NMR experiments of peptides P1 and P2…………..…........S31

11. References……………………………………………………………………………....S34

12. 1H NMR and MALDI-TOF/TOF spectra of peptides………………………………….S35

Electronic Supplementary Material (ESI) for ChemComm.This journal is © The Royal Society of Chemistry 2015

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1. ORTEP diagrams of peptides

Peptide P1:

A)

B)

Figure S1: A) ORTEP diagram of hybrid peptide Boc-(LUdγF)2-OEt (P1) containingα, β-

unsaturated γ-phenylalanine residues (represented by green color atoms) where ellipsoids are

drawn to 70% probability. Hydrogen bonds are represented in dotted lines. Only polar H-atoms

were shown for clarity. (CCDC number 1063358). B) View of Crystal packing forP1along b-

axis.

S3

Peptide P3:

Figure S2: ORTEP diagram of hybrid peptide Boc-(LUγF)2-OEt(P3)containing γ4-phenylalanine

residues (represented by green color atoms) where ellipsoids are drawn to 70% probability. Two

molecules of P3 present in an asymmetric unit.Hydrogen bonds are represented in dotted lines.

Only polar H-atoms were shown for clarity. (CCDC number 1060827).

S4

Figure S3: ORTEP diagram of peptide Boc-(LUγF)2-OEt(P3)(showing one molecule) where

ellipsoids are drawn to 70% probability. Hydrogen bonds are represented in dotted lines. Only

polar H-atoms were shown for clarity.

S5

Peptide P4:

Figure S4: ORTEP diagram of peptide Boc-(LUγA)2-OEt(P4)containing γ4-alanine residues

(represented by green color atoms) where ellipsoids are drawn to 69% probability. One ethanol

solvent molecule present in the asymmetric unit. Hydrogen bonds are represented in dotted lines.

Only polar H-atoms are shown for clarity. (CCDC number 1060826).

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2. H-bond Parameters

Table S1: Peptide P1 [Boc-LUdγFLUdγF-OEt]

i) Intramolecular H-bond parameters

Donor

(D)

Acceptor

(A)

D….A

(Å)

DH….A

(Å)

NH….O

(deg)

N4 O2 2.944 2.235 139.70

N6 O5 2.853 2.055 153.98

ii) Intermolecular H-bond parameters

Donor

(D)

Acceptor

(A)

D….A

(Å)

DH….A

(Å)

NH….O

(deg)

N1 O4§ 2.931 2.160 148.05

N2 O3§ 3.063 2.238 160.90

N5 O7† 2.934 2.075 176.72

O3 N2‡ 3.063 2.238 160.90

O4 N1‡ 2.931 2.160 148.05

O7 N5* 2.934 2.075 176.72

Symmetry transformations used to generate equivalent atoms:

§ 1.5-x, 1/2+y, 2-z;† 1.5-x, 1/2+y, 1-z; ‡ 1.5-x, 1/2+y, 2-z;* 1.5-x, 1/2+y, 1-z

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Table S2: Peptide P3[Boc-LUγFLUγF-OEt]

i) Intramolecular H-bond parameters for two molecules A and B in the asymmetric

unit

Acceptor

(A)

Donor

(D)

D….A

(Å)

DH….A

(Å)

NH….O

(deg)

O2 (A) N19 (A) 3.08 2.35 142.8

O3 (A) N16 (A) 2.95 2.12 163.5

O4 (A) N18 (A) 2.99 2.14 169.5

O5 (A) N17 (A) 2.84 2.04 153

O11 (B) N26 (B) 2.99 2.25 143.9

O12 (B) N23 (B) 2.92 2.07 164.8

O13 (B) N24(B) 3.04 2.20 165

O14 (B) N25 (B) 2.84 2.03 157.4

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ii) Intermolecular H-bond parameters for two molecules A and B in the asymmetric

unit

Donor

(D)

Acceptor

(A)

D….A

(Å)

DH….A

(Å)

NH….O

(deg)

Molecule A

N14 O16* 2.90 2.153 161

N15 O19* 3.00 2.068 169

O00B O21# 2.70 - -

O8† O21# 2.97 - -

O7 O20 2.84 - -

O7 N20 (B) 2.87 2.04 161

Molecule B

N20 O7 (A) 2.87 2.04 161

N21 O20 2.99 2.14 167

O16 O19 2.84 - -

N14 (A)‡ O16 2.90 2.07 161


*x, y, -1+z; # -1+x, y, z; † 1+x, y, z; ‡ x, y, 1+z

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Table S3: Peptide P4 [Boc-LUγALUγA-OEt]

i) Intramolecular Hydrogen bond parameters

Acceptor

(A)

Donor

(D)

D….A

(Å)

DH….A

(Å)

NH….O

(deg)

O9 N3 2.92 2.162 146.95

O8 N4 2.94 2.0841 171.6

O7 N5 2.93 2.102 160.68

O6 N6 3.00 2.209 153.93

ii) Intermolecular Hydrogen bond parameters

Acceptor

(A)

Donor

(D)

D….A

(Å)

DH….A

(Å)

NH….O

(deg)

N1 O4 * 2.80 1.95 168

O4 N1 † 2.80 1.95 168

O4 ‡ O10 # 2.88 2.14 165


* 1+x, 1+y, 1+z; † -1+x, -1+y, -1+z; # x, -1+y, z; ‡ x, 1+y, z

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3. Torsion angles of hybrid peptides

Table S4: Torsion angles of peptides P1, P4 and P6.

Pept. Resd. φ θ1 θ2 ψ ω

P1

Leu1 -95 - - -46 -163

Aib2 -60 - - -45 -174

dγF3 -129 18 -178 178 -178

Leu4 -48 - - 128 177

Aib5 68 - - 11 173

dγF6 -122 118 -178 -10 -

P4

Leu1 -53 - - -40±2 -175±2

Aib2 -57 - - -34 -174

γF3 -127±1 52 61 -114 -175±1

Leu4 -55±2 - - -39 178

Aib5 -59 - - -25 -173±1

γF6 -104 172±4 176/-161 98±15 -

P6

Leu1 -46 - - -41 -177

Aib2 -54 - - -37 -173

γA3 -132 55 59 -122 -169

Leu4 -62 - - -37 -178

Aib5 -67 - - -12 -177

γA6 -121 74 82 172 -

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4. Crystallographic Information

Crystal structure analysis of Boc-L-U-dγF-L-U-dγF-OEt (P1): Crystals of peptide were

grown by slow evaporation from a solution of methanol/toluene (1:1). A single crystal (0.21 ×

0.08 × 0.1 mm) was mounted on loop with a small amount of the paraffin oil. The X-ray data

were collected at 100K temperature on a Bruker APEX(II) DUO CCD diffractometer using Mo

Kα radiation (λ = 0.71073 Ǻ), ω-scans (2θ = 52.52 ), for a total of 14074 independent reflections.

Space group C2, a = 35.352(11), b = 10.430(3), c = 17.876(5), β = 107.549(5), V = 6285 (3) Ǻ3,

Monoclinic C, Z = 4 for chemical formula C49H72N6O9 (C7H8), with one molecule in asymmetric

unit; ρcalcd = 1.037 gcm-3, µ = 0.070 mm-1, F(000) = 2120, Rint = 0.0506. The structure was

obtained by direct methods using SHELXS-97.1 The final R value was 0.0542 (wR2 = 0.1280)

14074 observed reflections (F0 ≥ 4σ( |F0| )) and 589 variables, S = 0.832. The largest difference

peak and hole were 0.228 and -0.256 eǺ3, respectively.

There is some partially occupied solvent molecule also present in the asymmetric unit. A

significant amount of time was invested in identifying and refining the disordered molecule.

Option SQUEEZE of program PLATON2 was used to correct the diffraction data for diffuse

scattering effects and to identify the solvent molecule. PLATON calculated the upper limit of

volume that can be occupied by the solvent to be 1740 Å 3, or 27.7% of the unit cell volume. The

program calculated 118 electrons in the unit cell for the diffuse species. No data are given for the

diffusely scattering species. Outputs of SQUEEZE report are appended in cif file P1.

Crystal structure analysis of Boc-L-U-γF-L-U-γF-OEt (P3): Crystals of peptide were grown

by slow evaporation from a solution of methanol. A single crystal (0.12 × 0.09 × 0.1 mm) was

mounted on loop with a small amount of the paraffin oil. The X-ray data were collected at 100K

temperature on a Bruker APEX(II) DUO CCD diffractometer using Mo Kα radiation (λ =

0.71073 Ǻ), ω-scans (2θ = 56.7), for a total of 90866 independent reflections. Space group P 21,

a = 9.286 (3), b = 20.892 (7), c = 27.661 (9), β = 98.639(6), V = 5305 (3) Ǻ3, Monoclinic C, Z =

2 for chemical formula C49 H76 N6 O9(CH4 O, 3 O), with two molecule in asymmetric unit;

ρcalcd = 1.168 gcm-3, µ = 0.082 mm-1, F(000) = 1936, Rint = 0.1470. The structure was obtained

S12

by direct methods using SHELXS-97.1 The final R value was 0.1509 (wR2 = 0.3727) 26002

observed reflections (F0 ≥ 4σ(|F0|)) and 1224 variables, S = 1.642.

There are partially occupied solvent molecules along with peptide molecules.Option

SQUEEZE of program PLATON2 was used to correct the diffraction data for diffuse scattering

effects and to identify the solvent molecule. PLATON calculated the upper limit of volume that

can be occupied by the solvent to be 470 Å 3, or 8.8% of the unit cell volume. Outputs of

SQUEEZE report are appended in CIF file P4.This structure contains ethyl ester groups. Even

though this structure was collected at 100K the thermal parameters for one of the ethyl ester

group was very high. This was attempted to be modeled as disorder but this was not successful.

The conclusion is that this group is not well defined and thus was refined isotropically.

Crystal structure analysis of Boc-L-U-γA-L-U-γA-OEt (P4): Crystals of peptide were grown

by slow evaporation from a solution of ethanol. A single crystal (0.15 × 0.04 × 0.07 mm) was

mounted on loop with a small amount of the paraffin oil. The X-ray data were collected at 100K

temperature on a Bruker APEX(II) DUO CCD diffractometer using Mo Kα radiation (λ =

0.71073 Ǻ), ω-scans (2θ = 56.9), for a total of 7174 independent reflections. Space group P1, a =

9.7972 (4), b = 10.4809 (5), c = 12.3285 (6), β = 102.4740 (10), V = 1132 (9) Ǻ3, Triclinic, Z = 1

for chemical formula C37 H68 N6 O9 (C2 H6 O), with one molecule in asymmetric unit; ρcalcd =

1.129 gcm-3, µ = 0.081 mm-1, F (000) = 419, Rint = 0.0401. The structure was obtained by direct

methods using SHELXS-97.1 The final R value was 0.035 (wR2 = 0.1052) 7363 observed

reflections (F0 ≥ 4σ (|F0|)) and 514 variables, S = 0.908.

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5. General experimental details

All amino acids, triphenylphosphine, TFA, Ethyl bromoacetate, DCC, HOBt and LAH were

commercially available. DCM, DMF, ethyl acetate and petether(60-80 oC) have used after

distillation. THF was dried over sodium and distilled immediately prior to use. Column

chromatography was performed on silica gel (120-200 mesh). Final peptides were purified on

reverse phase HPLC (C18 column, MeOH/H2O 60:40-95:5 as gradient with flow rate 1.00

mL/min). 1H spectra were recorded on 500 MHz (or 13C on 125 MHz) and 400 MHz (or 13C on

100 MHz) using residual solvents as internal standards (CDCl3 δH 7.26 ppm, δC77.3 ppm).

Chemical shifts (δ) reported in ppm and coupling constants (J) reported in Hz.

NMR spectroscopy: All NMR studies were carried out by using a Bruker AVANCEIII-500 MHz

spectrometer. Resonance assignments were obtained by TOCSY and ROESY analysis. All two-

dimensional data were collected in phase-sensitive mode, by using the time-proportional phase

incrementation (TPPI) method. Sets of 1024 and 512 data points were used in the t2 and t1

dimensions, respectively. For TOCSY and ROESY analysis, 32 and 72 transients were collected,

respectively. A spectral width of 6007 Hz was used in both dimensions. A spin-lock time of 256

ms was used to obtain ROESY spectra. Zero-filling was carried out to finally yield a data set of 2

K × 1 K. A shifted square-sine-bell window was used before processing.

Circular dichroism (CD) spectroscopy:CD analysis was performed using cylindrical, jacketed

quartz cell (1 mm path length). Spectra were recorded with a spectral resolution of 0.05 nm, band

width 1 nm at a scan speed of 50nm/min and a response time 1 sec. All the spectra were

corrected for water solvent and are typically averaged over 3 scans.

S14

6. 2D NMR data of Peptides P1 and P2

Table S5: List of NOE’s observed in ROESY spectrum of P1

Residue H-atom Residue H-atom NOE observed

Leu (1) α CH Aib (2) NH Strong

Leu (1) NH Aib (2) NH Strong

Leu (1) NH Leu (1) αCH Medium

Leu (1) NH Leu (1) β CH Strong

Leu (1) NH Leu (1) γ CH Strong

Leu (1) NH Leu (1) δ CH Strong

Leu (1) α CH Aib (2) (CH3)2 Strong

Aib (2) NH dγF (3) NH Strong

Aib (2) NH Aib (2) (CH3)2 Strong

dγF (3) NH Aib (2) (CH3)2 Medium

dγF (3) αCH Leu (4) NH Strong

dγF (3) NH dγF (3) ω CH2 Strong

dγF (3) β CH dγF (3) ω CH2 Strong

dγF (3) γ CH dγF (3) Aromatic Strong

dγF (3) αCH dγF (3) γ CH Weak

Leu (4) NH Leu (4) β CH Medium

Leu (4) NH Leu (4) γ CH Weak

Aib (5) NH Leu (4) β CH Weak

Aib (5) NH Leu (4) αCH Strong

Aib (5) NH dγF (6) NH Strong


dγF (6) NH Aib (5) (CH3)2 Medium

dγF (6) NH dγF (6) ω CH2 Strong

dγF (6) NH dγF (6) αCH Strong

dγF (6) αCH dγF (6) ω CH2 Weak

dγF (6) αCH dγF (6) γCH Weak

dγF (6) αCH Aib (5) (CH3)2 Weak

dγF (6) β CH dγF (6) ω CH2 Strong

S15

6 α CH

3 α CH

3 β CH

6 β CH

6 NH

6 α CH3 α CH

2 NH3 β CH

6 β CH6 NH

Aromatic

5 NH4 NH3 NH

5 NH

2 NH

4 NH

3 NH

Aromatic

6 /66/5

3/2

4/3

Figure S5: Partial ROESY spectrum of Peptide P1.

6 α CH3 α CH

2 NH3 β CH

6 β CH6 NH

Aromatic

5 NH4 NH3 NH2 &5 CH3 & -O-CH2-CH3

Boc &2&5 (CH3)2

1&4 β, γ CH

3 & 6 ω CH2

1 α CH

4 α CH &-O-CH2

1 NH

6 γ CH

3 γ CH

3/3

3/36/6

5/5

5/44/4

2/2

2/1

5/4

2/1

3/26/5

6/6 6/6

6/6

6/5


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6 α CH3 α CH

2 NH3 β CH

6 β CH 5 NH4 NH

3 NH

3 & 6ω CH2

1 α CH

4 α CH &-O-CH2

1 NH

6 γ CH

3 γ CH

3/3

3/36/6

2/1

5/4

2/1

6/6

6/6

4/4

3/3

6/4

3/3 ortho Ar-H6/6

6/6

3/3

6/6 Ar-H

3/3 Ar-H

3β

CH

6 β

CH

3 N

H

6 NH

1 NH 6 γ CH3 γ CH


6 α CH3 α CH

2 NH3 β CH

6 β CH6 NH

Aromatic

5 NH4 NH3 NH

6 α CH

3 α CH

3 β CH

6 NH

5 NH

2 NH

4 NH

3 NH

Aromatic

6 β CH

6 α CH3 α CH

2 NH3 β CH

6 β CH6 NH

Aromatic

5 NH4 NH3 NH

2 &5 CH3 & -O-CH2-CH3

Boc &2&5 (CH3)2

1&4 β, γ CH

3 & 6 ω CH2

1 αCH

4 αCH &-O-CH2

1 NH

6 γ CH

3 γ CH


S17

Table S6: List of NOE’s observed in ROESY spectrum of P2

Residue H-atom Residue H-atom NOE observed

Leu (1) NH Aib (2) NH Strong


Leu (1) NH Aib (2) (CH3)2 Medium

Leu (1) NH Leu (1) β CH Strong

Leu (1) NH Leu (1) γ CH Weak

Aib(2) NH dγA (3) NH Weak

Aib(2) NH Aib (2) (CH3)2 Strong

Aib(2) (CH3)2 dγA (3) NH Strong

Aib(2) (CH3)2 dγA (3) α CH Weak

Aib(2) (CH3)2 dγA (3) γ CH Weak

dγA (3) NH dγA (3) CH3 Strong

dγA (3) β CH dγA (3) CH3 Strong

dγA (3) β CH dγA (6) CH3 Strong

dγA (3) α CH Leu (4) NH Strong

dγA (3) α CH dγA (3) NH Strong

dγA (3) α CH dγA (3) CH3 Medium

Leu (4) NH Leu (4) β CH Medium

Leu (4) NH Leu (4) γ CH Medium


Leu (4) α CH Leu (4) δ CH Strong

Aib (5) NH dγA (6) NH Medium


Aib (5) (CH3)2 dγA (6) NH Strong

Aib (5) (CH3)2 dγA (6) α CH Weak

Aib (5) (CH3)2 dγA (6) γ CH Strong

dγA (6) α CH dγA (6) NH medium

dgA (6) α CH dgA (6) CH3 Weak

dgA (6) NH dgA (6) CH3 Strong

dgA (6) β CH dgA (6) CH3 Strong

S18

6 α CH3 α CH2 & 5 NH’s

3 β CH6 β CH

4 NH3 NH6 NH

3 & 6 CH3 & -O-CH2-CH3

Boc

2&5 (CH3)2

1&4 β CH

6 /5

3/2

4/4

4/4 2/2

5/5

3/26 /5

3/2

3/2

3/6

3/3

6/6

3/3

6/6 6/6

3/3


6 α CH3 α CH2 & 5 NH’s

3 β CH6 β CH

4 NH3 NH6 NH

1 α CH

4 α CH&-O-CH2

3&6 γ CH’s

1 NH

4/5

1/2

1/2


S19

6 α CH3 α CH

2 & 5 NH’s

3 β CH6 β CH

4 NH3 NH6 NH

6 α CH

3 α CH

2 & 5 NH’s

3 β CH

4 NH

6 β CH

3 NH

6 NH

3/43/3

6/6

5/6 2/3

Figure S11: Partial TOCSY spectrum of Peptide P2.

6 α CH3 α CH

2 & 5 NH’s

3 β CH6 β CH

4 NH3 NH6 NH

6 α CH

3 α CH

3 β CH

4 NH

3 NH

6 NH

2 & 5 NH’s

6 β CH


S20

6 α CH3 α CH

2 & 5 NH’s

3 β CH6 β CH

4 NH3 NH6 NH

3 & 6 CH3 & -O-CH2-CH3

Boc

2&5 (CH3)2

1&4 β CH

1 & 4 CH3


3 & 6 CH3 & -O-CH2-CH3

Boc

2&5 (CH3)2

1&4 β CH

1 & 4 CH3 sidechain

1 α CH

4 α CH&-O-CH23&6 γ CH’s

1 NH


S21

iii)

Figure S15: A) Observed NOEs of the peptide P1 in the ROESY are schematically represented

by double headed arrows.

O

HN

HN

O HN

O

O

O

HN

O

NH

O

HN

OO

H

H

HH

Figure S15: B) Observed NOEs of the peptide P2 in the ROESY are schematically represented

by double headed arrows.

S22

Molecular Dynamics (MD): Model building and molecular dynamics simulation of P1 and P2

was carried out using Insight II (97.0) / Discover program on a Silicon Graphics Octane

workstation. The cvff force field with default parameters was used throughout the simulations.

Minimization’s were done first with steepest decent, followed by conjugate gradient methods for

a maximum of 1000 iterations each or RMS deviation of 0.001 kcal/mol, whichever was earlier.

The energy-minimized structures were then subjected to MD simulations. A number of inter

atomic distance constraints obtained from NMR data were used as restraints in the minimization

as well as MD runs. For MD runs, a temperature of 300 K was used. The molecules were

initially equilibrated for 50 ps and subsequently subjected to a 1 ns dynamics with a step size of

1 fs, sampling the trajectory at equal intervals of 10 ps. In trajectory 50 samples were generated

and the best structures were again energy minimized with above protocol and superimposed

these structures.

7. Synthesis and Characterization of Boc-dγF-OEt, Boc-dγA-OEt and Peptides P1-P4

Synthesis of Boc-dγX-OEt:3N-Boc-Amino aldehyde (10 mmol) was dissolved in 30 mL of dry

THF followed by Wittig ylide (11 mmol) was added at RT. Reaction mixture was stirred for

about 5 hrs at RT. Completion of the reaction was monitored by TLC. After completion, solvent

was evaporated and the crude product was purified by column chromatography using EtOAc/pet

ether to get (88%) of pure product.

(S,E)-ethyl 4-((tert-butoxycarbonyl)amino)-5-phenylpent-2-enoate:3White powder solid;Yield

88%; 1H NMR (400 MHz, CDCl3) δ 7.30-7.14 (m, 5H), 6.89 (dd, J=16 Hz, J=4 Hz, 1H), 5.84

(dd, J=16 Hz, J=2 Hz, 1H), 4.59 (b, 1H), 4.16 (q, J=7 Hz, 2H), 2.88 (m, 2H), 1.37 (s, 9H), 1.25

(t, J=7.1 Hz, 3H); 13CNMR (100 MHz, CDCl3) δ166.3, 155.2, 147.6, 136.4, 129.4, 128.6, 126.9,

S23

121.1, 79.9, 60.5, 52.3, 40.9, 28.3, 14.2; MALDI TOF/TOF m/z calculated value for C18H25NO4

[M+Na+] 342.1676 and observed 342.1657.

(S,E)-ethyl 4-((tert-butoxycarbonyl)amino)pent-2-enoate:3Colourless Oil; Yield 93%; 1H

NMR(400 MHz, CDCl3)δ 6.87-6.82 (dd, J = 16 Hz, J = 4 Hz, 1H), 5.89-5.85 (d, J = 16 Hz, 1H),

4.5 (br, 1H), 4.38 (br, 1H), 4.19-4.14(q, J= 8 Hz, 2H), 1.43 (s, 9H), 1.26-1.24 (m, 6H); 13C

NMR(100 MHz, CDCl3) 166.4, 154.9, 120.2, 79.8, 60.5, 47.0, 28.4, 20.4,

14.3;MALDI.TOF/TOFm/z Calculated value for C12H21NO4[M+Na]+ 266.1368 and

Observed266.1365.

Procedure for the synthesis of Boc-L-U-dγX-L-U-dγX-OEt:Tripeptide and hexapeptide

were prepared by conventional solution-phase fragment condensation strategy. Deprotections

were performed with trifloroacetic acid and saponification for N- and C-termini respectively.

Couplings were carried out using dicyclohexcarbodimide (DCC) and 1-hydroxybenzotriazole

(HOBt). Tripeptide was synthesised using 1+2 condensation strategy involving Boc-Leu-OH and

NH2-U-dgF-OEt. Hexapeptide was synthesized by 3+3 condensation strategy involving Boc-L-

U-dgX-COOH and NH2-L-U-dgX-OEt. All peptides were purified by RP-HPLC using

MeOH/H2O system.

Boc-LUdγF-OEt:White solid; yield 88%;1H NMR (400 MHz, CDCl3) δ 7.30 (bs, 1H), 7.29-

7.17 (m, 5H), 7.06 (d, J=7.8 Hz, 1H), 6.92 (dd, J = 15.8 Hz, J = 4.8 Hz, 1H), 6.47 (bs, 1H) 5.90

(d, J = 15.57 Hz, 1H), 4.90 (m, 2H), 4.16(q, 2H, J = 8 Hz), 2.91 (m, 2H) , 1.65 (m, 2H), 1.50-

1.40 (m, 16H), 1.26 (t, J= 7.1, 3H), 0.94(m, 6H);13C NMR (100 MHz, CDCl3) δ 173.53, 172.38,

147.05, 136.92, 129.35, 128.56, 126.83, 121.21, 80.82, 60.44, 57.54, 54.05, 53.53, 51.26, 40.62,

40.17, 34.03, 28.35, 25.61, 25.46, 25.03, 24.86, 23.03, 21.92, 14.33;HR-MSm/z calculated for

C28H43N3O6 [M+Na] + 540.3050, observed 540.3057.

Boc-LUdγA-OEt:1H NMR (400 MHz, CDCl3) δ 7.08 (d, J = 8 Hz, 1H), 6.88 (dd, J = 16 Hz, J

= 4 Hz, 1H), 6.48 (s, 1H), 5.92 (d, J = 16 Hz, 1H), 4.99 (bs, 1H), 4.65 (m, 1H), 4.16 (q, J = 8 Hz,

2H), 3.90 (m, 1H), 1.65 (m, 2H), 1.52 (s, 3H), 1.51 (s, 3H), 1.42 (s, 9H), 1.26 (m, 6H), 0.93 (m,

6H); 13CNMR (100 MHz, CDCl3) δ 173.5, 172.2, 166.7, 156.3, 149.1, 120.4, 80.8, 60.5, 57.5,

54.4, 46.1, 40.3, 28.4, 25.8, 24.9, 23.0, 22.0, 20.0, 14.4; HR-MS m/z value calculated for

C22H39N3O6 [M+Na+] 464.2736, observed 464.2740.

S24

Boc-L-U-dγF-L-U-dγF-OEt (P1): White color solid; Yield 50%; 1H NMR (500 MHz,

CDCl3) δ 7.12-7.02 (m, 11H), 6.66 (dd, J=15.6 Hz, J=5 Hz, 1H), 6.77 (m, 2H), 6.43 (b, 1H),

6.40 (s, 1H), 6.35 (s, 1H), 6.13 (d, J=15 Hz, 1H), 5.88 (dd, J=16 Hz, J=1 Hz, 1H), 4.88 (d, J=5

Hz, 1H), 4.83 (m, 1H), 4.76 (m, 1H), 4.07 (m, 2H), 3.78 (m, 1H), 2.91-2.73 (m, 4H), 1.66-1.42

(m, 6H), 1.40-1.16 (m, 25H), 0.86 (m, 12H);HR-MSm/z calculated value for C49H72N6O9

[M+Na+] 911.5253, observed 912.5266.

Boc-L-U-dγA-L-U-dγA-OEt (P2): White color solid; Yield 72%; 1H NMR (400 MHz,

CDCl3) δ 7.09 (d, J = 8 Hz, 1H), 7.01 (d, J = 8 Hz, 1H), 6.93 (d, J = 4 Hz, 1H), 6.89 (d, J = 4 Hz,

1H), 6.80 (d, J = 4 Hz, 1H), 6.76 (d, J = 4 Hz, 1H), 6.72 (d, J = 8 Hz, 1H), 6.58 (s, 1H), 6.57 (s,

1H), 6.14 (d, J = 16 Hz, 1H), 5.93 (d, J = 16 Hz, 1H), 5.08 (d, J = 4 Hz, 1H), 4.70-4.60 (m, 2H),

4.20-4.12 (m, 4H), 3.92-3.87 (m, 1H), 1.74-1.47 (m, 22H), 1.44 (s, 9H), 1.29-1.21 (m, 11H),

0.98-0.91 (m, 15H); HR-MSm/zcalculated value for C37H64N6O9 [M+Na+] 759.4632, observed

759.4623.

Transformation of β-miniature peptides into their saturated analogs of 10/12-

helices through catalytic hydrogenation:Boc-L-U-dγX-L-U-dγX-OEt (0.10 mmol, 100

S25

mg) was dissolved in EtOH (4 mL), and was treated with 20 mg of 20% Pd/C. The hydrogen gas

was supplied through balloon. The reaction mixture was stirred under hydrogen atmosphere for

about 5 h. The completion of the reaction was monitored by MALDI-TOF/TOF and RP-HPLC.

After the completion of reaction, the reaction mixture was diluted with EtOH (10 mL) and it was

filtered through sintered funnel using celite bed and celite bed was washed with EtOH (3 × 15

mL). The filtrate was evaporated under vacuum to get white crystalline pure product.

Boc-L-U-γF-L-U-γF-OEt (P3):White color solid; Yield 90%; 1H NMR (500 MHz, CDCl3) δ

7.92 (bs, 1H), 7.62 (bs, 1H), 7.40 (d, J = 15 Hz), 7.29-7.10 (m, 11H), 6.92-6.90 (m, 2H), 6.74-

6.63 (m, 2H), 5.53 (bs, 1H), 4.36-4.27 (m, 1H), 4.12-4.04 (m, 3H), 3.89-3.80 (m, 2H), 2.96-2.64

(m, 4H), 2.53-2.35 (m, 5H), 2.23-2.17 (m, 3H), 1.94-1.85 (m, 3H), 1.72-1.67 (m, 2H), 1.57 (s,

3H), 1.49 (s, 9H), 1.42 (s, 3H), 1.30 (s, 3H), 1.22 (t, J = 5 Hz, 3H), 1.02 (s, 3H), 0.98-0.88 (m,

12H); HR-MSm/z calculated value for C49H76N6O9 [M+Na+] 915.5571, observed 915.5574.

Boc-L-U-γA-L-U-γA-OEt (P4): White color solid; Yield 95%; 1H NMR (400 MHz, CDCl3)

δ8.03 (s, 1H), 7.15 (s, 1H), 7.08 (s, 1H), 6.83 (d, J = 10 Hz, 1H), 6.63 (s, 1H), 5.10 (d, J = 5 Hz,

1H), 4.13-4.06 (m, 2H), 4.03-3.86 (m, 5H), 2.55-2.34 (m, 4H), 2.16-2.13 (m, 1H), 2.03-1.97 (m,

1H), 1.86-1.73 (m, 10H), 1.60 (s, 3H), 1.53 (s, 3H), 1.48 (s, 9H), 1.44 (s, 3H), 1.37 (s, 3H), 1.23

(t, J = 5 Hz), 1.14 (d, J = 5 Hz, 3H), 1.04 (d, J = 5 Hz, 3H), 1.00-0.90 (m, 14H); HR-MS m/z

calculated value for C37H68N6O9 [M+Na+] 763.4945, observed 763.4940.

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9. CD spectra of peptides

200 220 240 260 280 300

-5x104

-4x104

-3x104

-2x104

-1x104

0

1x104

2x104

Mo

l.E

llip

(d

eg

.cm

2/d

mo

l)

Wavelength (nm)

Figure S16: CD spectrum of peptide P1 in methanol (c 0.2 mM).

S27

200 220 240 260 280 300-8.0x10

3

-6.0x103

-4.0x103

-2.0x103

0.0

2.0x103

Mo

l.E

llip

(d

eg

.cm

2/d

mo

l)

Wavelength (nm)


200 220 240 260 280 300-4x10

4

-3x104

-2x104

-1x104

0

Mo

l.ell

ip.

(deg

.cm

2.d

mo

l-1)

Wavelength (nm)


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200 220 240 260 280 300-3.0x10

4

-2.5x104

-2.0x104

-1.5x104

-1.0x104

-5.0x103

0.0

Mo

l.e

llip

(d

eg

.cm

2.d

mo

l-1)

Wavelength (nm)


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10. HPLC traces of peptides

0 10 20 30 40

0.0

0.5

1.0

1.5

2.028.20 min

AU

Time (min)

Figure S20: HPLC trace of peptide P1.

0 10 20 30 40

0.00

0.02

0.04

0.06

0.08

0.10

0.12

0.14

0.16

AU

Time (min)

25.5 min


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0 5 10 15 20 25 30 35

0.0

5.0x10-2

1.0x10-1

1.5x10-1

2.0x10-1

2.5x10-1

3.0x10-1

3.5x10-1

AU

Time (min)

25.9 min

Figure S22:HPLC trace of peptide P3.

0 10 20 30

0.00

0.05

0.10

0.15

AU

Time (min)

18.8 min


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11. Variable Temperature Study of Beta-meanders

Peptide P1:

Figure S24: Temperature dependent 1H NMR spectra of amide NH region of P1.

Table S7: Chemical shifts of amide NHs of peptide P1

Temperature

(0 °C) Leu1 Aib2 dγF3 Leu4 Aib5 dγF6

-40 5.34 6.73 6.99 7.59 6.44 7.47

-30 5.24 6.69 7.03 7.60 6.48 7.42

-20 5.15 6.62 7.06 7.47 6.47 7.38

-10 5.08 6.57 7.02 7.29 6.46 7.33

0 5.03 6.53 6.95 n.d 6.45 n.d

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10 4.98 6.50 6.90 n.d 6.45 n.d

20 4.96 6.47 6.85 6.71 6.44 n.d

30 4.93 6.46 6.82 6.56 6.44 n.d

40 4.89 6.43 6.78 6.43 6.43 7.08

dδ/dT

(ppb K-1)

-5.4

(± 0.49)

-3.8

(± 0.29)

-3.4

(± 0.56)

-16.0

(± 0.99)

-0.4

(± 0.17)

-4.9

(± 0.75)

-40 -20 0 20 40

5.0

5.5

6.0

6.5

7.0

7.5

Leu 1

Aib 2

dgA 3

Leu 4

Aib 5

dgA 6

Temperature (oC)

Ch

em

ical sh

ift

(pp

m)

Figure S25:Plot of amides chemical shift and the temperature. The slope dδ/dt for all NHs is given in the Table S5.

S33

Peptide P2:

Figure S26:Temperature dependent 1H NMR spectra of amide NH region of P3

Table S8: Chemical shifts of amide NHs of peptide P3

Temperature

(0 °C) Leu1 Aib2 dγA3 Leu4 Aib5 dγA6

-40 5.46 6.68 7.36 7.58 6.84 7.36

-30 5.36 6.66 7.29 7.55 6.67 7.35

-20 5.31 6.71 7.25 7.68 6.75 7.30

-10 5.22 6.69 7.19 7.54 6.69 7.25

0 5.15 6.63 7.13 7.38 6.67 7.19

10 5.09 6.58 7.06 7.11 6.62 7.13

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20 5.02 6.53 7.00 6.86 6.56 7.08

30 5.00 6.51 6.95 6.64 6.53 7.04

40 4.98 6.49 6.89 6.54 6.51 7.00

dδ/dT

(ppb K-1

) -6.2 (±0.4) -2.8 (±0.46) -5.9 (±0.09) -14.9 (±2.0) -3.7 (±0.5) -4.9 (±0.18)

-40 -20 0 20 40

5.0

5.5

6.0

6.5

7.0

7.5

8.0

Ch

em

ica

l s

hif

t (p

pm

)

Temperature (oC)

Leu 1

Aib 2

dgA 3

Leu 4

Aib 5

dgA 6

Figure S27: Plot of amides chemical shift and the temperature. The slope dδ/dt for all NHs is given in the Table S7.

12. References:

1. (a) SHELXS-97: G. M. Sheldrick, Acta Crystallogr. Sect A, 1990, 46, 467;(b) G. M.

Sheldrick, SHELXL-97, Universität Gӧttingen (Germany) 1997.

2. Spek, A. L. (1990) Acta. Cryst., A46, C34.

3. (a) S. M. Mali, A.Bandyopadhyay, S. V. Jadhav, M. GaneshKumar and H. N. Gopi,Org.

Biomol. Chem., 2011, 9, 6566;(b) A. Bandyopadhyay and H. N. Gopi, Org. Lett., 2012,

14, 2770.

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13. 1H NMR and MALDI-TOF/TOF spectra of compounds

S36

S37

Boc-LUdγF-OEt

S38

Boc-LUdγA-OEt

S39

S40

Peptide P1

S41

Peptide P2

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Peptide

P3

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Peptide P4

S44

Documents

Engineering polypeptide folding through trans … polypeptide folding through trans double bonds: Transformation ... Symmetry transformations used to generate equivalent atoms